JP3400991B2 - Rapid measurement of glycohemoglobin - Google Patents

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION Glycohemoglobin is a general term that refers to a series of hemoglobin minor components produced by the binding of various sugars (most commonly glucose) to a hemoglobin molecule. From the viewpoint of diabetes, the most important of these hemoglobin minor components is hemoglobin A _1c . It is formed by glucose binding to the N-terminal amino acid residue valine in one or both β chains of hemoglobin A [Goldstein, DE et al., Clin. Chem. 32: B64
-B70, 1986].

Human red blood cells are freely permeable to glucose. Glycohemoglobin is formed in each red blood cell from hemoglobin A (natural normal type) at a rate proportional to the ambient glucose concentration. Although this reaction is spontaneous and does not require enzyme catalysis, it is slow enough that only a small fraction of hemoglobin is modified during the life of red blood cells (120 days), which is irreversible. is there. as a result,
Glycohemoglobin is a measure of the weighted "moving" average of past blood glucose levels, with more recent blood glucose levels having a greater effect [Singer et al., Ann. Clin. Biochem. 26: 213-219, 1989].

Elevated levels of glycated hemoglobin are known to be associated with diabetes mellitus. Glycohemoglobin is present at levels of about 5% of total hemoglobin in non-diabetic patients, whereas diabetic patients exhibit 2-4 times this amount. Glycohemoglobin levels are relatively unaffected by short-term fluctuations in blood glucose levels and relatively accurately reflect the status of glycemic control in diabetic patients. The results are from the past 1
The average blood glucose concentration during the three months is shown. Glycohemoglobin measurements are used to assess the degree of glucose intolerance and control of diabetes mellitus in diabetic patients [Leste
r, Ann. Clin. Biochem. 26: 213-219, 1989; Kennedy
Et al., Br. Med. Bull. 45: 174-190, 1989; Flueckiger et al.,
J. Chromatogr. 429: 279-292, 1988; Goldstein, et al., C
lin. Chem. 32: B64-70, 1986; Mortensen, Dan. Med. Bu
ll. 32: 309-328, 1985; Goldstein et al., CRC Crit. Rev.
Clin. Lab. Sci. 21: 187-228, 1984, Peacock, J. Cli
n. Pathol. 37: 841-851, 1984; Miedema et al., Ann. Cli
n. Biochem. 21: 2-15, 1984; Mayer et al., Clin. Chem. Act.
a 127: 147-184, 1983; Gabbay, Med. Clin. North Am.
66: 1309-1315, 1982 ".

Glycohemoglobin is replaced by hemoglobin A _1c
Alternatively, there are various methods for measuring hemoglobin A1 or total glycohemoglobin (ion exchange chromatography, thiobarbituric acid method, isoelectric focusing method and affinity chromatographic analysis) [Cole, RA
Et al, Metabolism 27: 289-301, 1978; Nathan, DM Clin.
Chem. 27: 1261-1263, 1981; Moore, JC et al., Ann. Cli
n. Biochem. 23: 85-91, 1986]. In ion exchange chromatography, many glycated hemoglobin substances, including hemoglobin A _1c, are hemoglobin A _c at neutral pH.
Less positively charged than negatively charged and does not bind much to negatively charged resins [Rosenthal, PK et al., Am. J. Clin. Pathol. 75: 45-4
9, 1981; U.S. Pat.No. 4,407,961, U.S. Pat.No. 4,649,12
No. 2]. Several methods have been described for separating hemoglobin A _1c from the hemoglobin A _{1a + b} fraction [Go.
ldstein, DE et al., Diabetes 31: 70-78, 1982; Maquar
t, FX, etc., Clin. Chim. Acta 108: 329-332, 1980; Jon
es, MD. Et al, Hemoglobin 2: 53-58, 1978; Clarke, J.
T. et al., Diabete Metabol. 5: 293-296, 1979; Davis, J.
E. et al., Diabetes 27: 102-107, 1978; Cole, RA et al., M.
etabolism 27: 289-301, 1978; U.S. Pat.No. 4,389,491; Bi
o-Rad Laboratories, Hemoglobin A _1c Micro Column Tes
t Instruction Manual, March 1990]. However,
These methods have various drawbacks. Many of these methods use two buffers, the first buffer elutes unbound material from the ion exchange resin so as not to desorb the specifically bound material. A second buffer used at a different pH or ionic strength or containing a competitive inhibitor is required to elute the specifically bound material. temperature,
pH, ionic strength and column size influence the test results [Simon, M. et al., Diabetes 29: 467-474, 1980; S
chellekens, APM et al., Clin. Chem. 27: 94-99, 1981;
Castagnola, M. et al., J. Chromatogr. 272: 51-65, 198.
3]. Moreover, these methods require several different steps and several containers, most of which are not automated or only semi-automated.

Another limitation of these assays, depending on the method used, is the reversible intermediate glycoform "pre-hemoglobin-."
A _1c ”, which must be removed before performing the analysis [Goldstein, DE et al., Diabetes 31: 70-7.
8, 1982; Bunn, HF Diabetes 30: 613-617,1981; Nath
an, DM Clin. Chem. 27: 1261-1263, 1981; Mayer,
TK et al., Clin. Chim. Acta 127: 147-184, 1983; Healt
h and Public Policy Committee, American College of
Physicians Ann. Intern Med. 101: 710-713,1984; Nath
an, DM Clin. Chem. 27: 1261-1263, 1981]. High levels of fetal hemoglobin, sickle hemoglobin, and other rare conditions can interfere with this assay [Niejadlik, DC et al., J.
AMA 224: 1734-1736, 1973].

Another method of measuring glycated hemoglobin uses a specific affinity or binding agent that binds glycated hemoglobin. The next patent, U.S. Patent No. 4,
No. 200,435; No. 4,260,516; No. 4,274,978; No. 4,255,3
85; and 4,438,204, glycohemoglobin is measured using the affinity method or the allosteric properties of hemoglobin. German Patent No. 159569 describes sugar-binding proteins as affinity reagents.

Another affinity binding method is based on the specific complex formation between glycohemoglobin and boronic acid derivatives [Middle et al., Biochem. J. 209: 77.
1-779, 1983; Klenk et al., Clin. Chem. 28: 2088-2094, 19
82; Little et al., Clin. Chem. 32: 358-360, 1986, U.S. Pat. No. 4,269,605; U.S. Pat. No. 4,861,728; British Patent Application No. 2 206 411A; Isolab, Inc. Technical Publicati.
on: GlycAffin ^tm GHb, 1986; Forrest, RD, etc., Clin. Che
m. 34: 145-148, 1988]. The affinity binding method also detects glycohemoglobins in addition to HbA, which are proportional to more specific methods for HbA _1c , such as ion exchange chromatography [Little et al., Cl.
in. Chem. 32: 358-360, 1986]. Like the ion exchange and colorimetric assays for glycated hemoglobin, the affinity method also has its limitations. One of these limits is the need for two different buffers. The first buffer is cis
Elute the non-glycofraction without diol groups. The bound fraction, which is rich in glycated hemoglobin,
Elution with a second buffer containing a displacing agent that eliminates glycohemoglobin from the column, such as sugar alcohols. In addition, flow rates and column dimensions also limit the amount of hemoglobin bound to the affinity reagent.

There are various opinions about stable glycocosylated standards or calibration means or controls suitable for use in glycohemoglobin analysis to generate standard curves and determine analytical accuracy [Goldstein, DE et al., Clin. Ch
em. 32,10B: B64-B70, 1986; Franzini, C. et al., G. Ital.
Chim. Clin. 9: 187-192, 1984]. Some of these controls are nothing more than hemolysates of erythrocytes to which preservatives have been added [German Patent No. 150543; US Patent No. 3519572; British Patent No. 934461]. These materials are often lyophilized and reconstituted in the laboratory where they are used and used for days to weeks [Bio-Rad Laboratories, Hemoglobin A
_1c Micro Column Test Instruction Manual, March 199
0]. The stability of the material when done in this way is not well described. Some have attempted to form stable hemoglobin standards using cyanomethemoglobin; oxyhemoglobin-polyhydroxy compounds; and the carbon monoxide form of hemoglobin [German Patent 3311458; European Patent 72440; and Mosca. , A. et al., J. Clin.
Chem. Clin. Biochem. 23: 361-364, 1985]. Many laboratories control analytical accuracy by using locally generated controls (generally whole blood or packed red blood cell lysates made from normal and diabetic samples) [Goldst
ein, DE Diabetes 31: 70-78, 1982; Cole, RA etc. M
etabolism 27: 289-301, 1978; Simon, M. et al., Clin. Ch.
em. 28: 195-198, 1982; Parker, KM et al., Clin. Chem. 2
7: 669-672, 1981]. These controls must be stored under precisely controlled conditions to maintain stability [Wali
Nder, O. Clin. Chem. 28: 96-99 (1982)].

There remains a need for a glycohemoglobin assay that is easy to perform, is not inhibited, and is relatively insensitive to experimental variables such as pH and temperature. In addition, stable standards and controls for use in this type of analysis and methods of making the standards are also expected.

[0010]Summary of the invention The present inventionTechnologyIs the substrate-binding substance and group to be detected.
Substrate binding in samples containing non-binding substances
Method to detect and quantify the ratio or concentration of substances
It is about. More specifically, the present invention is
Sample containing substrate-binding substance and substrate-non-binding substance
To detect the ratio or concentration of substrate-binding substances in
It also relates to a rapid method for quantification.

According to the technique of the present invention, the absorbance of a sample containing a substrate-binding substance and a substrate non-binding substance is measured, and a solid substrate having a specific binding agent or an affinity agent attached thereto is added. The basic components in the binding reaction are the sample, the solid substrate with the binding agent attached, and a buffer solution compatible with the binding reaction.

After the binding reaction, the absorbance of the sample is measured using a suitable detector. Sample volume or relative activity is
It is inversely related to the amount of binding substance bound by the solid substrate.

Accordingly, one aspect of the technique relating to the present invention relates to a method for determining the presence and amount of a substrate-binding substance in a sample suspected of containing the substrate-binding substance, which method comprises The initial absorbance of a sample containing a reactive substance was measured, the substrate-binding substance was bound to a solid support having a binding agent specific to the binding substance attached, and the substrate-binding substance was removed or reduced. It involves measuring the absorbance of the sample and then calculating the ratio of substrate binding substances.

A preferred embodiment of the technique relating to the present invention relates to a method for determining the relative amount of glycated hemoglobin in a sample containing both glycated hemoglobin and non-glycated hemoglobin. A binder or affinity agent that specifically binds glycated hemoglobin is attached to a solid substrate, which is mixed and charged into a container adapted to separate the particles from the test solution. Two absorbance measurements are made on the test solution, the first one immediately after mixing the reagent / diluent with the test solution and the other absorbance measurement after glycated hemoglobin is substantially bound to the solid substrate. The analytical chemistry principle is based on the affinity binding of cis-diol compounds to 3-aminophenylboronic acid. The linear relationship between total glycated hemoglobin and hemoglobin A _1c allows normalization and recording of units corresponding to% hemoglobin A _1c .

[0015] The onset Ming, a method for producing a stable liquid glycated and non-glycated hemoglobin solutions for use in the measurement of glycated hemoglobin.

In yet another aspect, the present invention relates to a kit for use in measuring glycohemoglobin.

In yet another aspect, the technique related to the present invention
Surgery provides a method of determining glycohemoglobin ratios that is useful in diagnosing and managing diabetic patients.

[0018]Description of the drawings These and other objects, features and many advantages of the present invention
Will be better understood from the following detailed description with reference to the accompanying drawings.
Will be understood.

FIG. 1 is a schematic plan view of a test pack used in the preferred embodiment of the technique of the present invention. Details of the test pack are described in US Pat. No. 4,883,763, modified to include a porous filter. 2, Abbott Vision (TM) analyzer hand decision bovine azido - meth - a standard curve of glycated hemoglobin using hemoglobin calibration means. Figure 3 is a time course study of in vitro glycosylation of bovine hemoglobin as a function of glucose concentration. Figure 4 shows the stabilization of glycobovine azido-meth-hemoglobin by reduction with sodium cyanoborohydride, which was shown by stress for 24 hours at 45 ° C. FIG. 5 shows the comparative stability of glycated and reduced hemoglobin with non-glycated and non-reduced hemoglobin. Figure 6 shows the long-term stability of glycobovine azido-meth-hemoglobin calibrators stored at -20 ° C and 2-8 ° C. Calibration means A = 4.4 to 5.0% glycated hemoglobin (HbA _1c ); Assay means B = 12.4 to 1
4.0% HbA _1c ; test means C = 20.7 to 23.3%
HbA _1c . FIG. 7 shows a method and HP according to the technique of the present invention.
3 shows the correlation with the LC-Glyco Hb method. Correlation coefficient (CC) = 0.979. Figure 8 shows the correlation between the method according to the technique of the invention and the Isolab Glyc-Affin hemoglobin assay. Correlation coefficient (CC) = 0.9
91.

[0020]Detailed Description of the Invention The present inventionTechnologyThe main problem solved by
It was an automation of the method that required a single absorbance measurement.
The first measurement is the binding substance and non-binding substance in the sample.
Proportional to the total concentration of the substance, the other is the binding reaction
Ratio of either bound or free fractions after
It is an example. Both measurements from a single test solution into a single
There is a cause of difficulty in wanting to do it in the test container. This
This is a conventional column chromatograph using a self-contained reagent container.
Preferably automated without rough separation or buffer changes
It can be done in the device.

One aspect of the technique of the present invention provides a continuous (preferably automated) method for determining the ratio of substrate binding substance to total substrate binding substance in a sample. This method involves mixing a sample with a solid substrate, measuring the initial absorbance of a sample containing substrate-binding substance and non-substrate-binding substance, and measuring the absorbance of the sample from which the substrate-binding substance has been removed, It consists in calculating the ratio of substrate-binding substances. The solid substrate can be composed of particles to which a binder for the substrate-binding substance is attached. These particles are separated from the sample when measuring the absorbance. This can be done by several means,
One is to pass the mixture of sample and particles through a porous filter that holds these particles.

The substrate-binding substance can be any molecule that has an affinity for the binding agent. Binding substances of this kind include carbohydrates, proteins, nucleic acids, lipids and the like.
The binding substance can also be derived from animals, plants, bacteria, yeasts, protozoa, viruses, recombinantly produced substances and the like. When the substrate-binding substance is removed from the sample, a measurable change occurs in the sample, which can be detected by a device such as a spectrophotometer or a fluorometer. Preferably the measurable change is a change in the optical density of the sample as measured using a spectrophotometer. It will be readily appreciated that an amplification system is required to detect that the substrate binding material has been removed or partially removed from the sample. Amplification systems of this type are known in the art and include enzyme: substrate systems and the like.

One aspect of the technique relating to the present invention provides a method for determining the ratio or ratio of glycohemoglobin in a sample containing glyco and non-glycohemoglobin, which method comprises treating the sample to release glyco and non-glycohemoglobin. Treating and then contacting the solid substrate with the sample under conditions that result in complex formation between the glycohemoglobin and the solid substrate, and then measuring the initial absorbance associated with the total hemoglobin content of the sample to convert the glycohemoglobin to the solid substrate. It consists of separating this glycated hemoglobin from the sample by binding and then measuring the absorbance of the sample in which glycated hemoglobin is removed or in which glycated hemoglobin is significantly reduced. Techniques related to the present invention
The method of surgery includes, for example, RNA, other cis-diol-containing substances such as oligonucleotides, small physiological molecules such as catechol including D, L-dopa, epinephrine, norepinephrine, etc., such as citric acid, lactic acid. Can be used to detect α-hydroxycarboxylic acids such as, and other glycoproteins such as albumin. Even materials that do not have the required 1,2-cis-diol structure can often be readily derivatized with polyols, which can then be attached to the dihydroxyboryl moiety. For example, most nucleotides and nucleic acids are 25% in 0.5 M MOPS buffer (pH 5.5).
It can be treated with excess sorbitol and 1-ethyl-3 (3-dimethylaminopropyl) -carbodiimide (EDC) for 2 hours at 0 ° C to bind to sorbitol via the terminal phosphate [boronate ligand in biochemical separations, Publication 501, Amicon Corporation, 1981]. Deoxyribonucleotides and DNA not normally bound to boronates
Can be tightly coupled if a 3'or 5'terminal phosphate is available for polyol derivatization. The derivatized ligand can also be returned to its original form by treatment with phosphodiesterase if desired.
Using methyl-glucamine instead of sorbitol,
A chemically cleavable group can be formed. Alternatively,
Non-cis-diol containing molecules can also be attached to the dihydroxyboryl component by using cis-diol containing molecules as a binder, provided that the cis-diol containing molecules have an affinity for non-cis-diol containing molecules. I shall. This type of cis-diol and non-c
Examples of is-diol binding pairs include: Methyl-α-D-glucopyranoside: Concanavalin A; N
AD (P) ⁺ : glucose-6-phosphate dehydrogenase; ATP: hexokinase and the like.

The technique of the present invention for measuring glycated hemoglobin differs from the prior art in that the glycated hemoglobin is measured in a rapid continuous system which does not require elution of glycated hemoglobin from the solid support. Also, the technique relating to the present invention does not require the exchange of a buffer solution for analysis. Preferably the system is automated.

The present invention further provides well-defined and stable glyco standards and methods for making these standards for use as calibrators and controls in assays for measuring glycohemoglobin.

The principle of the technique relating to the present invention is based on the affinity binding of glycohemoglobin to 3-aminophenylboronic acid immobilized on small agarose beads,
Measured via dichroic absorbance at 553/628 nm.
An initial absorbance measurement proportional to total hemoglobin concentration is made after mixing the sample with the reagents and diluents. Complex formation between coplanar cis-diol groups of glycohemoglobin (eg HbA _1c ) and immobilized 3-aminophenylboronic acid occurs when the diluted sample is repeatedly mixed with boronate agarose. As a result, glycated hemoglobin is removed from the diluted sample. It is possible to calculate the bound Hb% by the final absorbance measurement that is proportional to the concentration of [total-glycohemoglobin], and standardize H using the prepared calibration curve.
Convert to bA _1c %.

As a first step, a sample (preferably a sample suspected of containing glycated hemoglobin, particularly preferably a whole blood sample) must be obtained. The following samples are preferred: whole blood anticoagulated with heparin or EDTA. However, samples collected in sodium citrate and sodium fluoride can also be used.
Prior to measuring glycated hemoglobin in blood samples, red blood cells need to be lysed. Lysis of red blood cells releases both glycated and non-glycated hemoglobin from the cells. Common cationic forms (eg cetyltrimethylammonium bromide); anionic forms (eg sodium dodecylsulfate and sodium deoxycholine); and neutral (eg saponin and octylphenoxypolyethoxyethanol) activators lyse red blood cells It is useful to let. About 0.02
Neutral activators in the concentration range of 5 to 0.5% by volume are preferred. Mechanical disruption, such as sonication and hypotonic lysis, are also effective ways to release hemoglobin from red blood cells.

To obtain long-term storage stability of the reagents, it is desirable to add small amounts of antimicrobial agents to the system, including solvents, antibiotics and poisons. Other biochemicals (eg KCN in the measurement of glycated hemoglobin) can also be introduced into the hemolyzed blood sample.

The sample is then treated with a binder or affinity agent specific for glycated hemoglobin to separate glycated hemoglobin from non-glycated hemoglobin. In one method, a sample of hemolyzed red blood cells containing glyco and non-glycohemoglobin is contacted with a solid substrate having a dihydroxyboryl moiety attached. In other embodiments, a solid support, e.g. HbA _1c to be specifically binding different binding agents or compatibilizers such as lecithin specifically binds to an antibody or HbA _1c binding.

The fixed support may be in the form of beads, resin or the like. In a particularly preferred embodiment, the solid support is in the form of individual particles or particulates. These particles may be composed of natural or synthetic polymeric materials, which may or may not be crosslinked, or may be chemically modified, as desired. Preferably the polymer is hydrophilic, eg polyacrylamide, or an agarose polyacrylamide copolymer or agarose eg as sold as Ultragel® or cellulose, eg cellulose derivatives, starch, dextran and cross-linked dextran. Polymers with various free hydroxyl groups, such as those sold under the registered trademarks Sephadex, Sepharose and Sephacryl. The diameter of the particles can vary over a wide range, but particles of 40 to 200 μm are preferred.

Particles of this nature serve as an immobilization substrate for the attachment of binders or affinity agents and can be easily used in containers. The container is one which causes the bond between the glycated hemoglobin and the fixed support and measures the absorbance. A preferred container is one in which the particles can be mixed with the test solution and which can be separated from the solution during the absorbance measurement. The most preferred container is US Pat.
It is an improved version of the solid phase analyzer or sample processing card as described in 4,883,763 (cited here for reference). The cylindrical bore in the mixing rib area houses the porous filter insert. The filter insert prevents the agarose particles from penetrating into the cuvette and allows the formation of an agarose packed bed through which the diluted sample flows. Precisely fill reagents and diluents in a peel cup. The dilution ratio is about 1:60. The dilution ratio is not important as this analysis does not measure mass / volume concentration.

Using this solid-phase analyzer, a specific binder for glycated hemoglobin is immobilized on the particles contained in the device. The particles are retained and immobilized. The sample is mixed with the solid substrate and the glycohemoglobin is captured and retained on the particles by the reagent on the particles. Non-glycated hemoglobin is not bound by the reagent and therefore remains free in solution.

Binders specific for glycated hemoglobin are:
US Pat. No. 4,269,605 (cited here for reference)
It may be composed of a dihydroxyboryl component as described in 1. This component is preferably phenyl or substituted phenylboronic acid, boric acid or other boronic acids such as ethaneboronic acid and 1-propaneboronic acid. The binder can be attached to the solid substrate by mechanical, physical or chemical means. Preferably, the ligand is directly covalently attached to the matrix. The binding agent should be attached to the substrate such that it does not desorb and liberate to hydroxyl boronate during subsequent reactions. In a preferred method, the affinity resin is carboxymethyl-agarose beads (CM- activated with m-aminophenylboronic acid using 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC).
It is prepared by reacting with Sepharose (registered trademark), Pharmacia. The reaction conditions are glycohemoglobin (G
In order to maximize the specific binding of Hb) and minimize the non-specific binding of non-glycated hemoglobin, it is chosen to give a product with the appropriate degree of substitution. Regardless of the dihydroxyboryl moiety chosen, it is necessary that the sugar moiety of glycohemoglobin be capable of binding to the dihydroxyboryl moiety to achieve the desired separation.

Alternatively, the specific binding agent may consist of an antibody which is coated, immobilized or covalently bound to the particles. Methods of obtaining antisera by immunization and recovery steps are generally known in the art. The antibody can also be a monoclonal antibody, the procedure of which is known in the art. Examples of monoclonal antibodies against HbA _1c are described in US Pat. No. 4,727,036 to Norres et al.

Other specific binding agents that can be coated, immobilized or covalently attached to particles include reagents specific for sugar moieties. These include α-D-, including concanavalin A, succinyl-con A and vicia fabba.
It includes lectins having specificity for glucose. Other lectins and their carbohydrate specificities are also well known and are available as free lectins or lectins bound to a solid support, for example Sepharose®-lectins. Other carbohydrate-specific supports include alumina gel, calcium phosphate gel, magnesium carbonate, magnesium silicate, silica gel.

Glyco and non-glycohemoglobin in a sample is a device for measuring the optical density (absorbance) of a solution,
It is preferably detected and quantified analytically using a spectrophotometer capable of measuring absorbance in the wavelength range of 340 to 633 nm. The device of the preferred embodiment is described in European Patent No. 0.
160 901 B1 (the disclosure of which is hereby incorporated by reference) for an automated Abbott Vision® analyzer.

The automated analyzer separates plasma from cells using the principle of two-dimensional centrifugation and measures and mixes fluids in a test pack. Centrifugal force generated by the rotating plate at the start of the test (rotating plate speed of 1800 rpm)
Moves each reagent in the test pack to one or more reagent measuring or holding chambers. At the same time, the sample is moved from the capillary or sample hole to the blood separation chamber. In the case of an anticoagulated and hemolyzed whole blood sample, centrifugal force causes the lysed erythrocyte stroma to settle, leaving the hemoglobin-containing sample at the top of the chamber. The rotating plate then continues to rotate as a series of rotations by the test pack holder occur.

The test pack holder rotates the test pack 90 ° counterclockwise to inject the buffer reagent and diluent buffer into the reaction chamber, and a portion of the sample is held in the sample holding chamber as shown in FIG. To inject. The test pack holder is then rotated 90 ° clockwise to move the liquid components of the reagent and diluent into the cuvette and move the sample to the measurement chamber. The test pack holder is then rotated 90 ° counterclockwise to move the measured sample into the reaction chamber where it mixes with the solid reagent and the liquid components of the reagent and diluent flowing out of the cuvette. The final 90 ° clockwise rotation of the test pack holder moves the diluted sample to the cuvette where the optics give an optical density at 553-628 nm for initial measurements and final measurements after multiple rotations of sample and reagent. To measure. Details of the Abbott Vision (R) Analyzer are described in Abbott Vision (R) Analyzer Manual, page 13.7. Other automated analyzers with similar specifications can also be used in the method.

[0039]Buffer The pH is cis-for the affinity column of the borate component.
Known to affect the binding of diol compounds
It High pH of buffer (greater than 9.6) and hemoglobin
At low pH values, whereas binding and stability of
Has increased non-specific binding. Non-specific binding is negatively charged
The boronate component and the sparse introduced by the phenyl ring.
Includes ionic effects associated with aqueous components. Used for analysis
When considering buffers to
Multiple hydroxy groups such as droxymethyl) aminomethane]
Since the buffer having a sil group binds to the boronate component
It should be avoided. Sorbitol, serine, ethanol
Amine and boric acid should also be avoided. Bole generated
A buffering agent that acts to strengthen the salt-diol complex is preferred.
Good Suitable buffers for this test system are about 7.5-
A buffer having a pKa in the range of 11.0. This range
Surrounding buffers are known in the art. 37 ℃ for analysis
At a pH range of about 7.8 to 9.6, more preferably about
P in the range of 8.5 to 9.2, particularly preferably 9 to 9.2
To maintain H, it has a pKa of about 8.5-9.2
Buffers are more preferred. Amine seems to strengthen the complex
Acts and thus for example glycine, morpholine, H
Buffers such as EPES or ammonium salts, for example
Or an additive like piperidine can help the binding
Is advantageous to. To maintain pH in preferred embodiments
The buffer used is 2-aminoethyl sulfonic acid (ta
Urin), 20 to 50 mM (preferably 25 m)
It has a pKa of 9.06 at the concentration of M).

Middle, FA et al., Biochem. J. 209: 771-77.
9 (1983) and “Boronate Ligands in Biochemical Separation, Publication 501, Amicon Corporation (1
981) ”describes the use of divalent cations, primarily Mg ²⁺ derived from MgCl ₂ to eliminate the repulsive force between the negatively charged immobilized boronate and the negatively charged ligand. The technique relating to the present invention is also used for this purpose as Mg ²⁺
To use. However, the techniques relating to the present invention may preferably use MgSO ₄ instead of MgCl ₂ to operate the invention optimally. The preferred concentration of MgSO ₄ is about 10-5.
00 mM, more preferably 50 to 200 mM, and particularly preferably about 100 to 150 mM. 3 for hemoglobin
Not very stable at 7 ° C and elevated pH. When the analysis is carried out in the presence of chloride, a decrease in absorbance of about 8% occurs in the absence of boronate agarose. This is due to the combination of precipitation and adsorption on the plastic surface of the test pack. A value of 8% reduction in absorbance is about twice the signal expected for a sample with normal glycohemoglobin levels without non-specific loss of hemoglobin from solution. In other words, the background is about twice the signal magnitude. MgSO instead of MgCl ₂
It was found that using ₄ reduced this loss by about 3%.

The inclusion of gelatin (fish scales) and polyvinylpyrrolidone (PVP) in the buffer further stabilized the hemoglobin.

In combination with MgSO ₄ , this gives a loss of about 1
% To decrease. The data from this experiment are shown in Table 1. Other protein stabilizers such as casein, gelatin from other sources, albumin and water-soluble polymers can be expected to give similar results.

[0043]

[Table 1]

[0044]analysis For the purpose of analysis, a known amount of solid substrate (10-200 μl
Range, typically 65 μl) in dry tablets or wet suspension
Supplied with an appropriate amount of buffer solution as either suspension.
The buffer solution adjusts the pH of the reaction to about 8. upon addition of the blood sample.
PKa suitable for maintaining a constant value in the range of 5-9.2
Containing 20 to 50 mM of the compound. About 10
500 mM, more preferably about 50-200 mM, especially
Preferably in buffer at a constant concentration of about 100-150 mM
Mg⁺⁺Salt was added to add GHb and boron
Eliminates the electrostatic repulsion between the two. Hemoglobin precipitation
Stabilizers and antimicrobial preservatives to prevent
Can be included in. Analysis temperature is about 24-39 ℃,
More preferably in the range of about 30-37 ℃, about 37 ℃
Is most preferred.

The blood sample is first treated with heparin to prevent clotting and with saponin to lyse the red blood cells. The sample is then centrifuged and a portion of the sample is roughly mixed with resin and buffer. Separate the diluted sample from the resin and obtain the initial absorbance of the diluted sample. The diluted sample is then mixed vigorously with the resin until the glycohemoglobin is almost bound to the solid substrate. The resin is taken out and the final absorbance is measured. The absorbance data is processed to calculate the relative amount or ratio of bound hemoglobin. Glycohemoglobin ratios are determined from a standard curve of bound hemoglobin to% glycated hemoglobin. A standard curve is constructed by the same procedure with a calibrated sample of glycated hemoglobin of known ratio. A known% glycated hemoglobin control is used to regularly test or confirm the accuracy of the standard curve.

[0046]Data organization of glycated hemoglobin Create a standard curve and measure unknown samples from this curve.
To do so, the analyzer performs the following steps. Whole hemog
Initial absorbance measurement value (A_i) And (Go
Total-unbound) Final absorbance measurement proportional to hemoglobin
(A_f) And are measured and stored. Bound hemoglobin%
The measured value of (% B) is calculated by the following formula: % B = 100 × (A_i-A_f) / A_i Calculate using. This bond% is calculated for the following two effects
Must be corrected: 1. Some degree of binding is the initial measurement
Occurs before. 2. Make a final measurement before reaching equilibrium. So
As a result, the measured% binding was affected by the total hemoglobin concentration.
Receive a sound. The% measured binding is consistent with the decrease in hemoglobin concentration.
Increase to. Therefore, the correction combination% (% D) is
Constants (E and I) and initial absorbance measurements (A_i) And
It is obtained by adjusting the% measurement binding: % D =% B−E × (A_i-I) Where I is the "normal" level at a concentration of about 13 g / dl.
Equivalent to the initial absorbance measurement value obtained for the moglobin sample
Good This parameter depends on the actual measurement wavelength and the test pack.
Dilution and the cuvette in the test pack
It depends on the road length. One of two different numbers for I
Whether the sample is a calibrator / control or a patient sample
Select according to whether there is. Dilution and path length are
It is adjusted during the manufacture of black. Actual measurement wavelength is different for each device
It changes slightly. Therefore, a unique pair of I
Used for storage. E is the% binding as a function of hemoglobin concentration.
Experimentally by keeping the% glycosylation constant
Is a constant obtained from. One of two different numbers for E
If the sample is a calibrator / control or patient sample
Is selected according to

The linear standard curve,% HbA _1c by using a predetermined value of is calculated from the regression analysis of the per% D and% HbA _1c calibration _{unit:% HbA 1c = (m ×} % D) + B [ Formula Where m is the slope and B is the Y-axis intercept].

Example: Standard curve:% HbA _1c = (0.8741 ×% D) +0.996 E = 7.7 × 10 ^-4 I = 1500

[0049]

[Table 2]

The standard curve is plotted in FIG. According to the conversion method using this data, it is possible to accurately measure% HbA _1c even for a sample having a hemoglobin concentration that greatly changes. One potential advantage is that a limited volume of sample (eg neonatal) can be diluted and still be analyzed precisely.

[0051]Calibration sample / control Glycohemog for use in glycated hemoglobin analysis
Robin calibration sample and / or control is preferably human
Alternatively, it is a liquid material made from animal blood. 1
In the specific example, the glycated hemoglobin material is bovine red blood cells?
Is created from Glycohemoglobin calibration sample is preferred
Are low, medium and high levels of glycoazide-
Met-hemoglobin (Example 3) preferably in reduced form
In a buffered solution at a pH of about 5-9, more preferably 6-8.
Contained in. Glycohemoglobin contained in calibration sample
Material level of total glycated hemoglobin (TGHb)
Ratio or hemoglobin (HbA_1c) Any of the ratio
Of hemoglobin in the sample.
Glycohemoglobin or hemoglobin for total amount
A_1cIs the amount of. Low level calibration sample is about 0-10% TG
Hb or 0-8% HbA_1c, More preferably about 2-7%
TGHb or 3-6% HbA_1cAnd particularly preferably 4 to 5
% TGHb or HbA_1cIs the range. Mid-level calibration test
The charge is about 10-27% TGHb or 8-16% HbA_1c,
More preferably 13 to 21% TGHb, ie 10 to 15%
HbA_1cParticularly preferably about 15-18% TGHb, ie
11-13% HbA_1cCan be in the range of. High water
The quasi-calibration sample is about 22-44% TGHb or 16-30
% HbA_1c, More preferably 26-36% TGHb, ie
18-25% HbA_1c, Particularly preferably about 27-30%
TGHb ie 19-21% HbA_1cCan be in the range of
it can. 45% TG for low, medium and high calibration samples
Hb or 30% HbA_1cGreater than, more preferably
About 45-60% TGHb or 30-40% HbA_1cNo Gu
Stock solution of glycated hemoglobin material in the lico% range
Formulated from a mixture with a non-glycol-reduced hemoglobin solution
can do. Very little non-glycated hemoglobin solution
The amount may include glycosides.

The stability of the calibrated sample and the control is such that when stored at about 8 ° C. or less, a stability of greater than about 10 months, more preferably in the range of 10-14 months, particularly preferably at least 14 months. Should have

The control is prepared in the same way as the calibration sample. The control level is preferably normal for glycated hemoglobin (4 to
6% HbA _1c ) and elevated (greater than 8% HbA _1c ) physiological levels are selected to reflect.

In one embodiment of the invention, the stable hemoglobin solution is made up of azido-meth-hemoglobin. Azido-meth-hemoglobin can be made from lipid-free hemoglobin released from washed red blood cells by hypotonic shock. The source of red blood cells is human or animal (preferably mammal), more preferably bovine red blood cells. Hemoglobin is oxidized to meth-hemoglobin by an oxidizing agent such as potassium ferricyanide or sodium nitrate. The met-hemoglobin is then contacted with an azide salt (such as sodium azide) to convert it to azido-meth-hemoglobin.

Hemoglobin in red blood cells is glycosylated in the presence of glucose by a non-enzymatic process. First, an irreversible reaction takes place between glucose and the N-terminal valine of the hemoglobin β chain, producing a labile aldimine or Schiff base. This rapid equilibrium is followed by a slow Amadori rearrangement, forming a stable ketoamine bond of hemoglobin A _1c . The measurement of hemoglobin A _1c is a useful index for long-term diabetes management.

[0056] In this onset Ming embodiment, stable glycated hemoglobin solutions azido - is created from hemoglobin - methemoglobin. A non-enzymatic hemoglobin reaction was used for in vitro synthesis of stable liquid glycated hemoglobin calibration samples and control materials. Azide from human or bovine erythrocytes
Metho-hemoglobin was incubated with various concentrations of glucose, preferably at 37 ° C for several days. Glucose molecules form amine groups and Schiff base materials on hemoglobin. After removal of unreacted glucose, unstable Schiff base materials are reduced with mild reducing agents (eg sodium cyanoborohydride), catalytic hydrides in the presence of catalysts such as palladium, sodium borohydride, etc. Stabilized by reduction of. The completion of the reduction can be tested by heat-stressing the reduced glycoazide-meth-hemoglobin at elevated temperature (eg about 45 ° C). Reduction by-products were removed by gel filtration or dialysis.

The concentration of glucose added to the azido-meth-hemoglobin to produce glycohemoglobin is about 50-1000 mM, preferably about 100-500 m.
M, particularly preferably in the range of about 150-300 mM. Glucose is incubated with azido-meth-hemoglobin for about 1-10 days or until the desired% glycosylation is reached.

In yet another embodiment, a cyano group can be used in place of the azido group. Cyano-meth-hemoglobin and reduced glycocyano-meth-hemoglobin also form stable hemoglobin and glycated hemoglobin solutions. Methods for making azido-meth-hemoglobin and reduced glycoazido-meth-hemoglobin can be used to make cyano-meth-hemoglobin and reduced glycocyano-meth-hemoglobin. Cyanide, such as sodium cyanide, is used in this method in place of the azide salt.

In yet another embodiment, carbonyl groups can be used in place of azido groups. Carbonyl-hemoglobin and reduced glycocarbonyl-hemoglobin also form stable hemoglobin and glycated hemoglobin solutions. A method of making carbonyl-hemoglobin involves blowing carbon monoxide gas into an oxy-hemoglobin solution prior to glycation. In this case, potassium ferricyanide or other oxidizing agent is not needed. This is because oxy-hemoglobin is a preferred precursor for carbonyl hemoglobin shaping. Defoamers are required to minimize foaming during the carbon monoxide blowing process. The resulting carbonyl-hemoglobin material is
It is glycosylated and treated similarly to azido-meth-hemoglobin or cyano-meth-hemoglobin.

[0060]kit Furthermore, the present invention provides a sample (preferably glyco and non-glycan).
Glycohemoglobus of blood sample containing cohemoglobin)
A test kit for use in analyzing bottle content
Also includes. This test kit contains the following components:
(1) having a pH of about 6-8 (preferably 7.7),
A binder for lycohemoglobin (preferably dihydro)
A xyboryl component, more preferably 3-aminophenylbom
Suspension of particles with rhoic acid attached and Mg²⁺With source
Buffer reagent solution and (2) the pH of the reaction mixture to 37
C. at about 7.8-9.6, more preferably 8.5-9.
5, particularly preferably in the range of 9 to 9.2.
And a buffering agent of about 7.5-11, preferably 8.5-9.
Diluent buffer solution with pKa of 2. Particles used for testing
Test kit components may be separate or
Can be supplied in combination or can be premixed into a single
It can also be supplied as a single solution mixture.

In another embodiment, the affinity particles have anti-glycohemoglobin antibody or lectin attached as a binder.

More preferably, the buffered reagent solution comprises dihydroxyboryl-derived particles and about 10-500 mM MgS.
O ₄ , particularly preferably about 100-150 mM MgSO 4.
Contains ₄ . The buffered reagent solution may also contain antimicrobial and / or preservatives such as gentamicin, sodium azide and the like.

Particularly preferably, the buffer reagent solution has a volume of 8.4 m.
M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and 150 mM MgS
Containing a suspension of 3-aminophenylboronic acid-derivatized agarose bead particles in a buffered reagent solution (pH 7.7) containing O ₄ , 0.4 g / l gentamicin sulfate and 0.05% sodium azide. To do. More preferably the diluting buffer solution is further eg gelatin,
It contains a protein stabilizer such as albumin, casein, etc., preferably fish scale gelatin, in a concentration of about 0.5 to 5%, more preferably 1 to 2%, particularly preferably 1.65%, and for example polyvinylpyrrolidone ( PVP) (K
Value 90) containing a water-soluble polymer in a concentration of about 0.5-5%, more preferably 1-2%, particularly preferably 1.65%, and further antimicrobials such as gentamicin and sodium azide. It also contains agents and preservatives. Preferably, about 190 μl of buffered reagent solution containing about 65 μl of precipitated hydrated beads is added to about 4-7 μl.
Used for each sample.

The buffered reagent solution and the diluent buffered solution can be packaged separately, in combination as two reagents or as a single reagent, more preferably as two reagents. In a preferred embodiment, the buffer reagent and diluent buffer are provided in a format such as a test pack. Preferably each test pack contains:
8.4 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and 150 mM
3-aminophenylboronic acid-derivatized agarose bead particles (3.5 mg, dry) in a buffer solution (pH 7.7) containing MgSO ₄ of 0.4 g / l of gentamicin sulfate and 0.05% sodium azide. Approximately 190 μl of buffer reagent containing (by weight) suspension. Diluent buffer is approximately 140 μl of buffer solution (pH 9.6) containing stabilizer and antimicrobial at the following concentrations: 9
4.2 mM taurine (2-aminoethylsulfonic acid), 1.65% fish scale gelatin, 1.65% polyvinylpyrrolidone (K value 90), 0.4 g / l gentamicin sulfate and 0.05%. Sodium azide.

Boronate-derived agarose tends to aggregate at high pH when used under final analytical conditions.
This makes material handling and dispensing difficult. To solve this problem, the reagent system is provided in a sealed 2-chamber device within the test pack. One chamber contains the boronate-agarose in low pH buffer (buffer reagent), the other chamber contains the diluent buffer solution. The contents of the two compartments are combined only at the time the assay is performed, at which point the pH of the boronate agarose is raised to the final assay pH. One of ordinary skill in the art will recognize that each component can be added to the sample individually or in combination, either sequentially or simultaneously. In a preferred embodiment, the buffer reagent solution and the diluent buffer solution are added to the sample simultaneously.
In another preferred embodiment, the buffer reagent solution is added after the diluent is added to the sample.

Preferably, the kit further comprises, as a third separate component, a lysing agent for the sample, such as a capillary containing dried saponin.

Calibration samples and controls can be packaged separately or packaged in a kit or supplied in a test kit. The calibration sample is preferably about 0-8%, 8-
16% and 16 to 30% HbA _1c reduction Gurikoajido having glycation levels in the range of - meth - containing hemoglobin. The comparison preferably contains reduced glycoazide-meth-hemoglobin with glycation levels in normal and abnormal patient ranges.

[0068]

【Example】Example 1 Creating a boronate agarose Agarose, a CM Sepha from Pharmacia
After washing Loin® CL6B with distilled water, 1
00 mM MES (2-N-morpholinoethane sulfone
Acid) buffer (pH 4.7). 5 agarose
Suspend to 0% solids, then cool to 0 ° C under reduced pressure
did. 66 mM of mAPBA (3-aminophenylboro
Acid), cooled to 0 ° C. under reduced pressure, then agar
Added to the loin. 800 mM EDC (1-ethyl-
3- (3-dimethylaminopropyl) carbodiimide salt
Acid salt) was made and added to the reaction mixture. This mixture
React under reduced pressure at 0 ° C for 1 hour, then 4M
The reaction was quenched with sodium acetate. mAPBA invitation
The derivatized agarose was sequentially treated with 100 mM acetic acid and 50 mM Na.
OH / 1M NaCl, 100 mM acetic acid, 50 mM Na
Wash with OH / 1M NaCl followed by distilled water. Wash
The purified mAPBA-derivatized agarose is then added to HEPES
It was washed with buffer and suspended to about 55% solids. Last
Check the pH of the mixture and adjust to a pH of 7.7 if necessary
did. Blank test according to the method in Example 2
Perform a calibration curve using the above particles in the test pack.
I made it. To obtain an accurate calibration curve,% solids of the particles
It can be adjusted by dilution or addition of particles.

[0069]Example 2 Abbott Vision Exam Pack and Abbott Vision
Glyco-Hb method using a gas analyzer Remove the vision test pack as shown in Figure 1 from the refrigerator.
put out. Warm test packs at room temperature for a minimum of 30 minutes
Let Dry saponin and heparin are filled and tested.
Insert the capillary containing the drug into the capillary slot of the test pack.
To enter. The calibration sample and control should be
And to the control hole directly using a dropping device. Test
Put the device into the analyzer and press the "Operation" button.

The analyzer then automatically performs the following steps:

[0071]

[Table 3]

[0072]Example 3 Preparation of calibration sample and reference material A. Preparation of azide-meth-hemoglobin (cow) 1. Plasma from bovine erythrocytes to 20 mM isotonic phosphate buffered saline
It was removed by washing with. 2. Freezing red blood cells with 0.01 M phosphate buffer
Knotting / thawing to dissolve by hypotonic shock
It 3. Molecular weight of hemoglobin up to 19.5 g / dl Hb
Ultrafiltration with 10,000 cut-off filters
Concentrated. The pH was adjusted to 7.2-7.8 with 2N NaOH.
Adjusted to. 4. 40 g / L of Aerosil 3 for lipid in Hb solution
80 (Van Waters). This mixture
The mixture was stirred at 4 ° C. for 1-18 hours. Mix to 4 ° C
Centrifuge at 4200 rpm for 10 minutes and
Globin was collected and filtered. 20 mM hemoglobin
Molecular weight 10,000 filter for phosphate buffered saline
It was diafiltered using. 5. Ferricyanation of hemoglobin in 1.3 x molar excess
Use potassium for 30-40 minutes at 2-8 ° C.
Oxidized to to-hemoglobin. 6. Metho-hemoglobin with 3 x molar excess of sodium
Add azide and add at a temperature of 2-8 ° C for 15-30 minutes.
Converted to zido-meth-hemoglobin. 7. This material is cut off with a molecular weight of 10,000
It was diafiltered with a filter. Adjust the pH of the solution to 7.3-7.5
Adjust and add antimicrobial agent. 8. Adjust the concentration to about 19.5g / dl hemoglobin
It was 9. This material was sterile filtered and stored at 2-8 ° C.

B. Preparation of glycated hemoglobin stock solution 1. Anhydrous glucose was added to azido-meth-hemoglobin and mixed for 30 minutes at 2-8 ° C to give a final glucose concentration of 250 mM. 2. The solution was incubated at 37 ° C until a glycation level of 30-40% was reached. 3. The solution is 2 to 8 with a filter having a molecular weight of 10,000.
Unreacted glucose (≦ 5 mg / dl) was removed by dialysis at 20 ° C. against 20 mM phosphate buffered saline. 4. Glycohemoglobin has a molecular weight of 10,000 to 30,
Concentrated to about 19.5 g / dl by diafiltration using a 000 cut-off filter. 5. The hemoglobin / glucose adduct was stabilized by reduction with the addition of a 10 × molar excess of sodium cyanoborohydride at 2-8 ° C. 6. The completion of the reduction was tested by heat treating the material at 45 ° C. Step 5 was repeated as needed. 7. Hemoglobin was dialyzed against phosphate buffer. 8. The hemoglobin concentration was adjusted to 19.5 g / dl. 9. The pH was adjusted to 7.4 and the solution was sterile filtered and stored at 2-8 ° C.

C. Preparation of Non-Glycohemoglobin Stock Solution This solution was made similar to the Glycohemoglobin stock solution except that glucose was not added.

D. The final calibration sample and control solutions were made by making a mixture of glyco and non-glyco stock solutions.

[0076]Example 4 Optimization of the method to glycosylate hemoglobin A liquid, stable glycoprotein was prepared using a non-enzymatic hemoglobin reaction.
For in vitro synthesis of hemoglobin calibration samples or controls
We have developed a method for According to the method in Example 3,
Fresh or frozen azido-meth prepared from bovine red blood cells
-Hemoglobin 250 at 37 ° C for various times
mM glucose (100 ml of 19.5 g / dl
For zido-meth-hemoglobin, 4.53 g of gluco
Culture). Unreacted glucose for gel filtration
Therefore, it is removed and the glycoazide-meth-hemoglobin is removed.
Reduction with excess sodium cyanoborohydride. Reduction by-product
Removed by dialysis against 20 mM phosphate buffered saline
The total glycohemoglobin% with Iso-lab Gly.
It was analyzed by the c-Affin affinity column method. result
Is shown in Table 3.

[0077]

[Table 4]

[0078]

[Table 5]

Human hemoglobin was similarly glycosylated and the results are shown in Table 4. Using cryo-stored human hemoglobin, a maximum% total glycosylation of 63% was observed after about 144 hours, at which time human hemoglobin began to aggregate (Table 4).

A time-course comparison of glucose concentration to total glycosylation and bovine hemoglobin showed two important factors in the glycosylation method (FIG. 3). First, maximum glycosylation%
Increased with increasing glucose concentration, and secondly the rate of% glycation was accelerated at higher glucose concentrations. Both factors contribute to optimizing the glycosylation process.

To prove that heat treatment at 45 ° C. for 24 hours is a valid indicator of the effect of sodium cyanoborohydride (NaCNBH ₃ ), high levels of calibrated samples were tested at various concentrations at 45 ° C. Incubated with NaCNBH ₃ . Various amounts of NaCNBH ₃ were added to the high level of the calibration sample based on the heme concentration in the calibration sample,
The final concentrations shown in the table were obtained. Each solution is divided into two parts, one is kept at 45 ℃ for 24 hours, the other is 2-8 ℃
Stored in. The data from this experiment are shown in Table 5 and FIG.
Shown in. This experiment shows the effect of NaCNBH ₃ reduction on the stabilization of glycated hemoglobin when present in appropriate amounts and the effect of 45 ° C. heat treatment used as a monitor or control for NaCNBH ₃ reduction.

[0082]

[Table 6]

To demonstrate that sodium cyanoborohydride reduction of glycobovine azido-meth-hemoglobin increases the stability of hemoglobin (Hb) compared to the stability of non-glyco non-reduced azido-meth-hemoglobin. , About 17.5 g / dl Hb per glycated reduced sample
And 1 part each at a concentration of 18.1 g / dl for the non-glyco non-reduced sample was incubated at 45 ° C and the same concentration of control was stored at 2-8 ° C. Each sample was periodically taken at a predetermined time interval for 34 days, and the hemoglobin concentration was measured. The results in Table 6 and Figure 5 show that the glycosylated reduced bovine azido-meth-hemoglobin is more stable than the non-glyco non-reduced form.

The long-term stability of the reduced glycobovine azido-meth-hemoglobin calibrator was determined at -20 ° C and 2-8 ° C.
It was measured by heat retention at. One copy of each standard for a total of 30
HbA _1c % was tested daily for 9 days. The results in Table 7 and Figure 6 show that the calibrator is stable for long term storage at either -20 ° C or 2-8 ° C. No adverse effect was observed.

[0085]

[Table 7]

[0086]

[Table 8]

[0087]Example 5 Expected value Using the method of the present invention, the glycated hemoglobin reference range is
4.1-5.7 Standardized% HbA_1cWas determined to be
It was This range includes 101 apparently healthy individuals (41 females).
, 60 men) were tested and confirmed. Special reference range
It may vary depending on the specific patient population.

[0088]Example 6 Reproducibility The present inventionTechnologyReproducibility of method by one analysis
2 levels of glycolysis in 4 repeats over 5 days using the device
Determined by field testing the hemoglobin comparison
did. Total average and coefficient of variation (% CV) from variation analysis
I calculated. All% CVs are based on diurnal and diurnal changes
Is the sum of.

[0089]

[Table 9]

[0090]Example 7 accuracy The present invention for determining the% Glyco Hb testTechnology
The accuracy of the method, this test and HPLC glycated hemoglobin
Analysis or Isolab Glyc Affin analysis
By comparison with (total glycohemoglobin by affinity)
Determined by field tests on human whole blood samples. % Green
A method and a method according to the invention for measuring cohemoglobin.
Correlation test between lycohemoglobin and HPLC method
It was

[0091] about the present invention to measure glycated hemoglobin
4.5 to 13.3% by implementing the method of technology in a hospital environment
Was performed in a hospital with 51 human whole blood samples spanning a range of glycated hemoglobin values of. The results are
It was correlated with the results obtained using the C-glycohemoglobin method.

All samples were analyzed in situ with a single sample. The correlation between the Vision Glyco Hb system and the HPLC Glycohemoglobin system is shown in FIG. Linear regression analysis gave the following results: Comparison: Vision Glyco Hb (Y-axis) vs. HPLC Glycohemoglobin (X-axis) Sample number = 51 Slope = 0.98 intercept = 0.33 Correlation coefficient = 0.983 Bias = 0.2 Sd line = 0.4 Sd difference = 0.4 t = 0.40 No statistically or clinically significant difference was observed.

Glycohemoglobin vs. Isolab Gl
The following correlations were observed using the method of the present invention for the ycAffin test: N = 51 slope = 0.652 intercept = 1.40 correlation coefficient = 0.991 bias = −1.6 Sd line = 0. .3 Sd difference = 1.3 vs. t = −8.97 unpaired t = −2.69 These data are plotted in FIG.

[0094]Example 8 Disturbance The following commonly prescribed agents interfere with the method of the invention.
Inspect and test whole blood samples containing additive substances for harm
did. The next substance at a given concentration is a certain level of Hb
It showed less than 10% interference for the sample containing A1c:       Test substance              Concentration          % HbA _1c     Endogenous substances       Bilirubin 30mg / dl 4.7       Glucose 800mg / dl 4.5       Triglyceride 1300mg / dl 4.8     Drugs prescribed for type II diabetes       Chlorpropamide 75 mg / dl 5.5       Glipizide 50 mg / dl 5.5       Glyburide 50mg / dl 5.5       Tolbutamide 100 mg / dl 5.8

[0095] The following materials, to the present invention in the sample concentrations shown
It did not significantly interfere with the method of technology involved. Significant interference is defined as causing a difference greater than 10% in test results for samples in the normal range.

[0096]         Commonly prescribed drugs Tested concentrations     Acetaminophen (Tylenol) 30mg / dl     Acetylsalicylic acid (aspirin) 50ml / dl     Ascorbic acid (vitamin C) 10mg / dl     Caffeine 6mg / dl     Cefoxitin 2.5 mg / dl     Cimetidine (Tagamet) 7.5mg / dl     L-dopa 10mg / dl     Epinephrine (adrenaline) 0.1 mg / dl     Hydrochlorthiazide 5mg / dl     Isoniazid 2.5 mg / dl     Penicillin G 1000 units / dl     Phenytoin (dilantin) 8mg / dl     Salicylic acid 40mg / dl     Theophylline 8mg / dl     Warfarin (Coumadin) 10mg / dl

[0097]Example 9 Hemoglobin variant (abnormal hemoglobin) The affinity binding method of glycated hemoglobin is
Shows minimal interference by species [Middle, F.A., et al., Biochem.
 J. 209: 771-779, 1983; Abraham, E.C. et al., J.Lab. Cli
n. Med. 102: 187-197, 1983; Talwar, D. et al., Clin. Che
m. Acta 128: 61-67, 1983; Yatscoff, R.W. et al., Clin. C.
hem. 29: 543-545, 1983]. Hemoglobin F (fetus),
S and C represent the present inventionTechnologyThe test described in
It turned out not to harm. Similarly, do not interfere with other variants.
Seems to be.

[Brief description of drawings]

FIG. 1 is a schematic plan view of a test pack for use in a preferred embodiment of the technique of the present invention. For more information on the test pack, see U.S. Patent No. 4, modified to include a porous filter.
No. 883,763.

FIG. 2 is a standard curve of glycated hemoglobin using a bovine azido-meth-hemoglobin calibration means as determined by the method of the technique of the present invention on an Abbott Vision® analyzer.

FIG. 3 is a time course study of in vitro glycosylation of bovine hemoglobin as a function of glucose concentration.

FIG. 4 shows the stabilization of glycobovine azido-meth-hemoglobin by reduction with sodium cyanoborohydride as shown by stress at 45 ° C. for 24 hours.

FIG. 5 shows the comparative stability of glycated and reduced hemoglobin and non-glycated and non-reduced hemoglobin.

FIG. 6 shows the long-term stability of glycobovine azido-meth-hemoglobin calibrators stored at −20 ° C. and 2-8 ° C. Calibration means A = 4.4 to 5.0% glycated hemoglobin (HbA _1c ); Assay means B = 12.4 to 14.0%
HbA _1c ; test means C = 20.7 to 23.3% Hb
A _1c .

FIG. 7 shows the correlation between the method according to the invention and the HPLC-glycoHb method. Correlation coefficient (CC) = 0.97
9.

FIG. 8: Method and Isolab Gl according to the technique of the present invention
3 shows the correlation between yc-Affin hemoglobin analysis. Correlation coefficient (CC) = 0.991.

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Barbara The Ray England United States, Wisconsin 53211, Milwaukee, East Kenwood Boulevard 1808 (72) Inventor Mary Jeanie Anino United States, Illinois 6004, A Lynton Heights, North Highland Avenyu 2616 (56) References JP-A-59-183370 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 33/72

Claims (15)

(57) [Claims] 1. In preparing a stable glycohemoglobin solution useful as a calibration sample or control in the analysis of glycated hemoglobin: a. Washing red blood cells; b. Release hemoglobin from red blood cells into the solution; c. Removing lipids from the hemoglobin solution; d. Oxidize hemoglobin to meth-hemoglobin; e. Converting meth-hemoglobin to azido-meth-hemoglobin; f. A method for producing a stable glycohemoglobin solution, which comprises adding glucose to azido-meth-hemoglobin to produce glycoazide-meth-hemoglobin. 2. The red blood cell is a mammalian red blood cell.
The method described in. 3. The method of claim 1 wherein meth-hemoglobin is converted to azido-meth-hemoglobin using sodium azide. 4. The method of claim 1, further comprising reducing glycoazide-meth-hemoglobin with a reducing agent. 5. The method according to claim 4, wherein completion of the reduction is tested by a temperature rising heat treatment test. 6. In preparing a stable glycohemoglobin solution useful as a calibration sample or control in the analysis of glycated hemoglobin: a. Washing red blood cells; b. Release hemoglobin from red blood cells into the solution; c. Removing lipids from the hemoglobin solution; d. Converting hemoglobin to carbonyl-hemoglobin; e. A method for producing a stable glycohemoglobin solution, which comprises adding glucose to carbonyl-hemoglobin to produce glycocarbonyl-hemoglobin. 7. The method of claim 6, further comprising reducing glycocarbonyl-hemoglobin with a reducing agent. 8. Making a stable non-glycated hemoglobin solution useful as a calibration sample or control in the analysis of glycated hemoglobin: a. Washing red blood cells; b. Release hemoglobin from red blood cells into the solution; c. Removing lipids from the hemoglobin solution; d. Converting oxy-hemoglobin to carbonyl-hemoglobin, or oxidizing hemoglobin to meth-hemoglobin; e. A method for producing a stable non-glycohemoglobin solution, which comprises converting meth-hemoglobin to azido-meth-hemoglobin. 9. In preparing a stable non-glycated hemoglobin solution useful as a calibration sample or control in the analysis of glycated hemoglobin: a. Washing red blood cells; b. Release hemoglobin from red blood cells into the solution; c. Removing lipids from the hemoglobin solution; d. A method for producing a stable non-glycohemoglobin solution, which comprises converting hemoglobin to carbonyl-hemoglobin. 10. A stable glycated hemoglobin solution which is useful as a calibration sample or a control in the analysis of glycated hemoglobin, which is characterized by consisting of glycated azido-meth-hemoglobin or glycated carbonyl-hemoglobin. 11. A stable glycohemoglobin solution which is useful as a calibration sample or control in the analysis of glycohemoglobin, which comprises reduced glycoazide-meth-hemoglobin or reduced glycocarbonyl-hemoglobin. 12. A stable non-glycohemoglobin solution useful as a calibration sample or control in the analysis of glycated hemoglobin, characterized in that it comprises azido-meth-hemoglobin or carbonyl-hemoglobin. 13. In a separately packaged kit of stable glycated hemoglobin standards for use in a method of determining the relative amount of glycated hemoglobin in a sample: a. Reducing Gurikoajido - methemoglobin - hemoglobin or reduced glycated carbonyl - solution containing with hemoglobin enable measurement within the total hemoglobin content in pairs to a range of 0 to 10% solution of total glycated hemoglobin and; b. Reducing Gurikoajido - methemoglobin - hemoglobin or reduced glycated carbonyl - solution containing with hemoglobin enable measurement within the range of 1 0 to 27% against the total hemoglobin content of the solution of all the glycated hemoglobin and; c. Reducing Gurikoajido - methemoglobin - hemoglobin or reduced glycated carbonyl - solution containing with hemoglobin enable measurement within the total hemoglobin content in pairs on 2 2-44% solution of total glycated hemoglobin and; d. A kit comprising a solution of azido-meth-hemoglobin or carbonyl-hemoglobin. 14. Each component comprises: a. Reducing Gurikoajido - methemoglobin - hemoglobin or reduced glycated carbonyl - solution containing with hemoglobin enable measurement within the total hemoglobin content in the pair and the 2-7% of the solution of all the glycated hemoglobin and; b. Reducing Gurikoajido - methemoglobin - hemoglobin or reduced glycated carbonyl - solution containing with hemoglobin enable measurement within the range of 1 3-21% against the total hemoglobin content of the solution of all the glycated hemoglobin and; c. Reducing Gurikoajido - methemoglobin - hemoglobin or reduced glycated carbonyl - solution containing with hemoglobin enable measurement within the total hemoglobin content in pairs on 2 6-36% solution of total glycated hemoglobin and; d. The kit according to claim 13, comprising a solution of azido-meth-hemoglobin or carbonyl-hemoglobin. 15. In a kit of stable glycated hemoglobin standards for use in a method of measuring the relative amount of glycated hemoglobin in a sample: a. Reducing Gurikoajido - methemoglobin - hemoglobin or reduced glycated carbonyl - solution containing with hemoglobin A _1c measurable percentage in the total hemoglobin content in the pair and the range of 4 to 6% hemoglobin solution and; b. Reducing Gurikoajido - methemoglobin - hemoglobin or reduced glycated carbonyl - kit, characterized in that comprising a solution containing with hemoglobin A1c total hemoglobin content in the pair to a high measurable proportions than 8% hemoglobin solution.